Making nanostructured materials from maize, milk and malacostraca

Nano-structured materials are used in electronics, diagnostics, therapeutics, smart packaging, energy management and textiles, areas critical for society and quality of life. However, their fabrication often places high demands on limited natural resources. Accordingly, renewable sources for the feedstocks used in their production are highly desirable. We demonstrate the use of readily available biopolymers derived from maize (zein), milk (casein) and malacostraca (crab-shell derived chitin) in conjunction with sacrificial templates, self-assembled monodisperse latex beads and anodized aluminium membranes, for producing robust surfaces coated with highly regular hyperporous networks or wire-like morphological features, respectively. The utility of this facile strategy for nano-structuring of biopolymers was demonstrated in a surface based-sensing application, where biotin-selective binding sites were generated in the zein-based nano-structured hyperporous network.

Sacrificial LB template surfaces were prepared using aqueous solutions of monodispersed latex (polystyrene) beads [100 (LB1), 300 (LB3) or 800 nm (LB8)] which were drop-coated on functionalized Au/quartz or silicon wafer (Scheme 1-SI) and residual solvent (water) evaporated (Sect. 1.3.2 in SI). The morphologies of the latex bead-coated surfaces were examined by scanning electron microscopy (SEM), which revealed highly compact and uniform lattice-like arrangement of the beads with long-range uniformity (see also, Fig. 1A-C-SI). In the case of AAM, with well-defined cylindrical nanopores ( Fig. 1D-SI), membranes were placed directly on the substrate surfaces.
Nanostructured zein surfaces were prepared by drop-casting methanolic solutions of defatted-zein on functionalized Au/quartz or silicon wafer in the presence of sacrificial latex beads (Scheme 1-SI) or alumina membrane (Scheme 2-SI), or absence of a sacrificial template (Table 1-SI). After solvent evaporation under vacuum, the LBs and AAM sacrificial templates were selectively dissolved in toluene and aqueous HCl, respectively. SEM images of the zein films after extraction of the LB sacrificial templates showed long-range arrays (mm scale) of interconnected uniform spherical cavities with dimensions reflecting the size of the latex bead used (Figs. 1A-C, 2-SI). Interestingly, the magnitudes of the differences in the resonant frequencies corresponding to masses of the zein film prepared with and without LB on Au/quartz surfaces decreased with increased bead size ( www.nature.com/scientificreports/ This can be attributed to the larger cavities (lower polymer density) present in the films prepared with the larger beads.
In the case of zein films fabricated using AAM as a sacrificial template, SEM images revealed long-range arrays of 150 nm thick coatings of zein-based nanowire-like features (Fig. 1D). The thickness of the nanowires can be manipulated by using AAMs with different pore sizes ( Fig. 3B-SI). The lower mass of zein deposited in the presence of AAM (Table 2-SI), relative to the corresponding thin film (Fig. 1E), again demonstrated the lower densities arising from the spacing of the nanowires (Fig. 3A-SI).
We then moved on to investigate the impact of the nanostructured zein films on permeability with electrochemical impedance spectroscopy (EIS) using Fe(CN) 6 3− /Fe(CN) 6 4− as a redox couple. The real and imaginary components of the complex-plane impedance measure the diffusion of an electroactive redox couple through the porous biopolymer film reflecting. Impedance plot for zein films shows an arc at higher frequencies (Fig. 3) in contrast to the unhindered diffusion-controlled electron transfer reaction on a bare Au surface with a straight line inclined with a gradient of π/4 ( Fig. 4B-SI). The diameter of this arc is the measure of the charge transfer resistance (R ct ) and the diffusion afforded by the biopolymer film for the redox electron transfer reaction. The larger the diameter the greater the resistance to charge transfer or diffusion. The biopolymer film templated with 100 nm beads before extraction with toluene shows higher R ct (135.5 ± 4.3 kΩ) (Table 1, Fig. 3) than the  Table 1 www.nature.com/scientificreports/ unmodified gold surface (200 Ω) (Fig. 4B-SI). Upon extraction of the templated beads in toluene the R ct value is significantly reduced (68.5 ± 3.1 kΩ) owing to the diffusion of the redox couple via the pathway generated through the interconnected pores in the zein film (Fig. 1A). This behavior was observed for all LB-templated zein films (Table 1). Noticeably, 800 nm LB-templated zein films displayed even lower R ct values (41.7 ± 2.2 kΩ), attributed to the greater pore size enabling more effective diffusion of the redox couple. These decreasing R ct values reflect the more macroporous nature of the zein films obtained when using larger-sized LBs. Thickness of the Z-LB3 film was measured using profilometry and estimated to be 2 µm (Fig. 4C-SI), as compared to the   The zein nanowire-coated surfaces were more permeable than thin-film controls (Fig. 1E), as revealed by reduced R ct values when compared to zein provide access or channel for the diffusion for redox couple, though less permeable than the highly porous LB templated zein films ( Table 1). The stabilities of the LB-and AAMderived zein nanostructures were monitored after storage in PBS (pH 7.4) at 20 °C for 6 months (see Sect. 1.4-SI). No significant changes in the zein nanostructured surfaces were observed by SEM (Fig. 5-SI), highlighting the robust nature of these materials.
The potential for deploying zein nano-structured materials to enhance sensor performance through improved mass-transfer was examined by fabricating a series of biotin imprinted zein nanostructures on Au/quartz resonator surfaces. The performance of these surfaces was studied using a quartz crystal microbalance and flow injection analysis (FIA) and compared with their non-imprinted counterparts and non-nanostructured zein coatings (Fig. 6-SI). Selective biotin recognition was demonstrated, and the LB3-templated imprinted materials induced significant enhancements in sensitivity. The hierarchical imprinted material architectures produced by combining molecular imprinting with the use of sacrificial templates illustrated the potential for these materials in applications requiring efficient mass-transfer. It is important to note that the total binding is enhanced by the larger available surface area, which results from contributions for both the non-specific binding and the increased accessibility to sites selective for biotin.
The scope for nano-structuring other readily available biopolymers was examined by replacing zein with the milk protein casein and the crustacean (e.g. Malacostraca, crab) derived oligosaccharide chitosan. SEM revealed that the nano-structured surfaces prepared using sacrificial LB-template displayed features comparable to those obtained using zein (Figs. 7-and 8-SI). When using AAM as a template, chitosan nanowires were readily obtained ( Fig. 8B-SI), though casein nanowires did not survive the low pH used for the extraction of alumina membrane. RAIR spectra of the casein and chitosan nanostructures (Figs. 9-, 10-SI) showed the presence of -NH 2 , CONH 2 and OH functionalities groups comparable to those of the corresponding non-templated biopolymer films Ca-EtOH and CHI-AcOH. The permeability characteristics of the casein and chitosan nanostructured films reflected those obtained with zein (Table 1). However, the casein and chitosan films were not as stable as those produced with zein as evidenced by the deformation of the macroporous structure (Figs. 11-and 12-SI) after storage for 6 months. These observations were reflected in EIS permeability studies, which showed insignificant differences in R ct value for zein films after storage, whereas casein and chitosan films showed a sharp increase in R ct values after storage indicating the onset of deformation. (Table 3-SI). This is in agreement with the reported stability of the zein films to solvents and high ionic strengths 50 .
Nanostructured biopolymer films can be easily obtained using sacrificial templates and readily available and renewable biopolymer feedstocks. This facile bench-top method provides access to material morphologies and associated permeabilities that can be used to advantage in situations requiring efficient mass-transfer, as illustrated here by the introduction of hierarchical features into nanostructured zein films through biotin molecular imprinting and use for enhancing biotin detection using a quartz crystal microbalance. The capacity to tailor nanostructure characteristics through choice of sacrificial template and biopolymer opens for the use of biopolymer-based nanostructured materials in a range of surface-based technologies. Studies are underway to explore the broader application of these materials in surface-based sensors and as catalytic supports.  www.nature.com/scientificreports/